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Enterotoxigenic Escherichia coli (ETEC) are a major cause of diarrheal disease worldwide. Adhesion pili (or fimbriae), such as the CFA/I (colonization factor antigen I) organelles that enable ETEC to attach efficiently to the host intestinal tract epithelium, are critical virulence factors for initiation of infection. We characterized at single organelle level the intrinsic biomechanical properties and kinetics of individual CFA/I pili, demonstrating that weak external forces (7.5 pN) are sufficient to unwind the intact helical filament of this prototypical ETEC pilus and that it quickly regains its original structure when the force is removed. While the general relationship between exertion of force and an increase in the filament length for CFA/I pili associated with diarrheal disease is analogous to that of P-pili and type 1 pili, associated with urinary tract and other infections, the biomechanical properties of these different pili differ in key quantitative details. Unique features of CFA/I pili, including the significantly lower force required for unwinding, the higher extension speed at which the pili enter a dynamic range of unwinding, and the appearance of sudden force drops during unwinding can be attributed to morphological features of CFA/I pili including weak layer-to-layer interactions between subunits on adjacent turns of the helix, and the approximately horizontal orientation of pilin subunits with respect to the filament axis. Our results indicate that ETEC CFA/I pili are flexible organelles optimized to withstand harsh motion without breaking, resulting in continued attachment to the intestinal epithelium by the pathogenic bacteria that express these pili.
Escherichia coli are Gram-negative bacteria that are a normal constituent of the intestinal tract microbiota of humans and warm-blooded animals. Some pathogenic strains of enterotoxigenic E. coli (ETEC) cause diarrheal disease with severe symptoms and dehydration, leading to annual mortality of hundreds of thousand children below the age of five in developing countries 1. Our studies investigate the structural features of the archetype ETEC pilus, CFA/I, which promotes host/pathogen interactions, thereby initiating infection. The aim of this research is to reveal structural clues for the development of novel drugs against this critical ETEC virulence factor.
Adhesion pili are specialized to sustain attachment of bacterial cells under the environmental conditions surrounding their preferred host target tissue. For ETEC, this environment is the small intestine. Fluid in the small intestine contains a mixture of solutes, e.g., carbohydrates, peptides and lipids from ingested food, and secretions from the biliary tree and pancreas that contain various digestive enzymes, e.g., electrolytes and pH-regulating substances. The intestinal wall has longitudinal and circular smooth muscle layers that provide mixing and propulsive movement of this chyme for efficient digestion. Contractile rings interspaced along the intestine segment the chyme into compartments and create a circular motion of the fluid. The propulsive movement results in forward fluid movement, and simulations have shown that the contraction phase of the peristaltic reflex generates pressure, shear stress, and a reverse vortex-like flow of the chyme 2. Bacteria are therefore exposed to a harsh environment which requires sophisticated adhesive tools, i.e. the pili, to maintain stable adhesion.
CFA/I pili that adhere to the intestinal epithelium are approximately 1 μm long helical filaments with a diameter of 7.4 nm and 3.17 pilin subunits per turn of the helix 3. The major pilin subunit, CfaB, is an approximately cylindrical protein that comprises seven beta strands in an IgG-like structure 4. The N-terminal strand of one subunit fills a hydrophobic groove of the preceding subunit, thereby producing strong non-covalent interactions along the filament. This helical filament architecture is also seen in P-pili and type 1 pili expressed on uropathogenic E. coli (UPEC) that infect the bladder and may reach the upper urinary tract and kidneys 5; 6, as well as type 3 pili expressed by Klebsiella pneumoniae that cause respiratory tract infections 7, and E. coli S pili, which are correlated to neonatal meningitis and urinary tract infections 8. Adhesion pili assembled via this “donor strand exchange” mechanism 9 provide effective damping against external shear forces by unwinding of their quaternary structure while leaving the tertiary structure intact. This property provides distribution of the shear forces amongst several attached pili and thereby increases the adhesion lifetime 10; 11. Thus, extension of pili by unwinding of the helical filament allows for motion without breaking the binding structure. Since CFA/I pili are similar in architecture to UPEC-expressed pili 3 we believed that CFA/I pili also were capable of unwinding their quaternary structure and also regaining their original structure after exposure to force, in a similar way to what has previously been found for other pili 8; 12; 13.
In this work, we used data from three methodologies in order to characterize and elucidate the function of CFA/I pili under force exposure. Force measuring optical tweezers (FMOT) were used to measure the force required to unwind an individual pilus at a single organelle level. Data were also collected to assess bond kinetics during unwinding. The interactions between adjacent layers responsible for pilus stability were modeled and analyzed in this study using the quaternary structure and orientation of subunits determined previously by a hybrid approach that combined crystallographic data with results from electron microscopy (EM)14. Finally, the unwinding and retraction biomechanics were modeled by Monte Carlo simulations and fitted to the data.
Our results strongly suggest that the force needed for pilus unwinding is a function of both subunit-subunit interactions and the pitch of the subunits. The limited subunit-subunit interactions as well as their horizontal subunit orientation relative to the normal direction of the applied force lead to the low forces required for CFA/I pili's filament unwinding. Thus, physical properties of the pilus filament facilitate the sustained adhesion of ETEC bacteria in the gut, and thereby facilitate initiation of diarrheal disease.
CFA/I pili are normally observed in both the wound and unwound state, as seen in the AFM and transmission EM images of cells expressing CFA/I pili shown in Fig. 1. The biomechanical properties of CFA/I pili were investigated with FMOT, to provide information on the force-extension relationship, bond opening rates, and detailed properties of intersubunit bonds. In order to avoid any effect of the distal tip and the adhesin, we bound a pilus non-specifically to a bead. Tensile stress was then applied by separating the bacterium-bead complex as described in the methods section. A typical unwinding response of an individual CFA/I pilus under steady-state conditions is shown in Fig. 2A. The extension response can be divided into three regions that represent different geometrical configurations of the pili. Region I represents elastic stretching of the quaternary structure constituting only a small fraction of the total elongation. At the microscopic level it represents straightening of the helix-like filament and an increase, without disruption of the layer-to-layer (LL) interactions, of the distance between the layers. Region II is entered at higher force where the layers are extended further and the interactions between subunits in adjacent layers are sequentially broken at a qualitatively constant threshold force, unwinding the helical filament. The sequential nature of the unwinding is caused by higher probability of breaking bonds adjacent to an already broken bond, in comparison to bonds that are surrounded by intact LL bonds 15. This unwinding occurs at a force of ~7.5 pN for CFA/I pili, during which a filament with a helical structural design has been transformed under force into a much longer open helical structure in which subunits are only connected head-to-tail via the N-terminal extension. In the experiment shown in Fig. 2A, the pilus detached from the bead at ~2.1 μm, resulting in a zero force response. A histogram of the unwinding forces for all investigated CFA/I pili, is shown in Fig. 2B. The average unwinding force, representing the strength of the LL interaction, was found to be 7.5 ± 1.5 pN (n = 88 distinct CFA/I pili). This is substantially lower than the average unwinding forces observed in the same experimental setup with S, P and type 1 pili expressed by UPEC, which unwind at 21 ± 2 pN, 28 ± 2 pN, and 30 ± 2 pN, respectively 8; 12.
The force required for the unwinding of CFA/I pili in Region II correlates with weak LL interactions between subunits. Our analyses of the crystal structure of CfaB modeled into the helical reconstruction CFA/I pili from EM data (Supplemental Figure) shows that the surface contact area in CFA/I pili is 710 Å 2, as compared with 1150 Å 2 for PapA in P-pili (Fig. 3). If the force required to pull apart adjacent layers of the pilus had increased linearly with increased surface area, our data would predict that the difference in force to unwind CFA/I pili would be only approximately 60 % less than that of P-pili. Instead, the measured decrease is 3.7-fold. To look at the interactions in more detail, the specific interactions between residues that produce LL bonds were examined. As CFA/I pili and P-pili are helical filaments with 3.17 and 3.28 subunits per turn, respectively, LL interactions could occur between subunits that are 2, 3, and 4 subunits farther along the helical axis. As shown in Fig. 3, results calculated by van der Waal's overlaps ≥ −4Å show that CFA/I pili have seven residue-residue interactions between subunits, whereas P-pili have 28 interactions. As seen in Table 1, supplementary material, in CFA/I pili all seven interactions are between the n and n+3 subunit, whereas in P-pili there are 11 interactions between n and n+3, and 17 interactions between n and n+4.
In addition to the specific residue-residue interactions discussed above, the orientation of the pilin subunits in the helical filament also appears to play a role in the force required for unwinding. The force available for unwinding the pilus filament is the component of force normal to the subunit-subunit interaction between layers. Thus, when the pulling direction is along the filament axis, the orientation of subunits with respect to this axis defines the proportion of the exerted force, tensile stress, available for unwinding. For CFA/I pili, the subunits are oriented only 4° from horizontal, resulting in almost all of the force contributing to unwinding, which requires 7.5 pN of force for steady-state unwinding. P-pili on the other hand are oriented 13° from horizontal and require a force of 28 pN 15. In contrast to the orientations of these subunits, the subunits of Hib pili are oriented 82° from horizontal. While FMOT experiments have not been performed on Hib pili from Haemophilus influenzae bacteria, in EM data these pili are never seen to unwind 16. These data indicate that the forces required for purification, including shearing of Hib pili from the bacteria using a blender, are not sufficient to unwind these filaments that have almost vertically-oriented subunits.
The fluctuations of the constant force level of CFA/I pili during LL unwinding are larger than the fluctuations that have been seen for other pili. As seen in Fig. 2, the force required for unwinding the LL interactions of CFA/I fluctuated on average ~2 pN about the mean, in comparison to ~1 pN for P- and S- pili. This fluctuation is not only larger, but also represents a much larger fraction of the force required for unwinding, 2 pN / 7.5 pN = 26% for CFA/I pili as compared with 1 pN / 28 pN = 4% for P-pili.
The higher force response, denoted region III in Fig. 2A, corresponds to elastic stretching of the pilus once it is already in its linearized form. A linearized pilus can suitably be described, since the units comprising a long filament are much smaller than the filament's contour length, as a semi-flexible polymer. However, subunits in a pilus are connected head-to-tail with a constraint regarding the degree of rotation, wherefore a free-jointed chain model does not acceptably reproduce the force-extension response. Instead, a pilus behaves more like a continuously flexible uniform rod, in which the units act together as a “worm-like chain” 17, as previous work has shown 10; 18 ENREF 18. The almost linearly increasing force seen in region III originates from the extension of a Worm–Like Chain (WLC) for a limited interval in proximity to its contour length. In Fig. 2A, the CFA/I pilus started its elastic response at ~2.0 μm with an almost linearly increasing force that suddenly dropped to zero at ~2.1 μm when the pilus detached from the bead. Force-extension curves in measurements with forces up to the highest forces that can be achieved at high precision with the current experimental method, 60-70 pN, always showed an approximately linear response in Region III.
It is possible to analyze the force-rewinding behavior of an individual pilus by reversing the direction of motion of the probing bead. An example of data from a pilus being unwound (gray) and contracted (black) is shown in Fig. 4. The two curves overlap, indicating that the measurement is in a steady-state regime 15. However, the rewinding curve shows a small dip in force, ~4 pN, at ~1.9 μm. Such dips are always observed for P and type 1 pili12 and originate from the lack of a nucleation kernel, i.e., there are no layers formed that can wind the subunits into the helix-like shape. Therefore, linearized pili require a certain amount of slack before an n and n+3 subunit are in close enough proximity to form a LL interaction. This particular behavior suggests that the CFA/I pili are assembled using a mechanism similar to that of P-pili.
To quantify the persistence length of the unwound CFA/I pili, a WLC was fitted (red dashed line in Fig. 4) to experimental data with Eq. (1). The persistence length, which is a measure of the resistance towards bending, was assessed to 4.5 ± 1.4 nm, which is slightly longer than for previously assessed values of P and type 1 pili, 3.2 ± 0.6 and 3.3 ± 1.6 nm respectively 10; 18.
We then sought a plausible explanation for the lack of an S-shaped transition, and therefore lack of a conformational change, when additional force was applied to CFA/I pili that were in region III of the curve. As seen in Fig. 5, in an extended conformation, the n to n+1 CfaB subunits are closely apposed, correlating with less flexibility than between PapA subunits The data support CFA/I pili adopting a more rigid structure than P, S, F1C, or type 1 pili by the end of region II. This more rigid fiber is capable of resisting the conformational change that is visualized as the S-shaped force-extension curve in region III seen in for P, S, F1C, and type 1 pili.
We then compared the architecture and connections between adjacent units of the major subunit CfaB in CFA/I pili to PapA in P-pili, to look more closely at the molecular details, and noticed both similarities and differences. A crystallographic structure of two adjacent CfaB subunits and two adjacent PapA subunits revealed a possible explanation of the transition that is observed in region III of P-pili unwinding but not in CFA/I pili. In Fig. 5. adjacent PapA subunits are moderately spaced with residues between subunits that can form a hinge, allowing flexibility between PapA subunits. Conversely, adjacent CfaB subunits are closely spaced, with no residues available to form a flexible hinge. This close apposition limits the ability of the subunits to rotate with respect to each other without steric clashes.
We calculated the persistence length, R, of the wound CFA/I helical rod morphology from electron micrographs. The variable curvature of a rod gives an indication of the rigidity of its structure, and thus the strength of the LL interactions. A micrograph of negatively stained CFA/I is shown in Fig. 1B. It is seen that the filaments have inherent flexibility along their length, rather than adopting a long, straight morphology. It was found that the average persistence length for CFA/I was 1.4 μm, whereas previous work indicated that P-pili are rather straight with a R of 8 μm 6. EM data also reveal the ease with which CFA/I pili unwind, as noted by the presence of unwound filaments in essentially all fields of view of CFA/I micrographs, in contrast to their relatively rare appearance in P-pili 6 and their total absence in Hib pili 16. These results are reproduced in our AFM imaging analysis of bacteria expressing CFA/I pili, as unwound pili are observed on some imaged cells (e.g. Fig. 1A).
The complete force-extension response was modeled by Monte Carlo methods, similar to the method presented in 18. The pili extension model included elastic stretching, a WLC model, and a two state energy landscape where the kinetics were described via a stochastic probability function weighted by the Arrhenius factor. A fit (black curve) of the model to an experimental force curve (gray curve) is shown in Fig. 6. Clearly, the model excellently reproduces the force-extension behavior of a CFA/I pilus.
A series of dynamic force spectroscopy (DFS) measurements is shown in Fig. 7. Each datapoint represents the unwinding force, i.e., force of region II, for a given elongation speed, L˙. In this study we performed DFS at 0.1, 1, 2, 4, 8, 16, and 32 μm/s. The horizontal solid line is a fit to the data points representing steady-state, defined as the region for which the force required to unwind the filament is not dependent on the elongation speed. The tilted solid line is a fit to F() = kBT/xATln(/th), where xAT is the bond length and th is the thermal elongation speed 15. In this equation the force required for unwinding increases as the elongation speed increases. As a result, the bond length can be derived from the inclination (slope) of the fitted curve. The intersection of the dashed lines corresponds to the corner velocity, *, which is the elongation speed that separates the steady-state regime from the dynamic regime. This was assessed to 1400 ± 200 nm/s.
It has been shown that pilus unwinding enhances bacterial adhesion to host cells by distributing the forces acting on a bacterium in a flow to several pili 11. It is also likely that this unwinding property has coevolved with the adhesin to optimize the adhesion lifetime of the bacterium 19. From the measurements presented here, it is concluded that CFA/I pili also can unwind and that the biomechanical model developed in refs 15; 20 describes the data well. Since the structure of these pili is principally the same as type 1 and P-pili, this was to be expected a priori. However, there were remarkable biophysical differences between CFA/I and the previously studied helix-like pili expressed. Mechanical parameters are summarized in Table 1. For example the unwinding force of S, P, F1C and type 1 pili are within 21-30 pN, i.e., ~3-4 times higher than CFA/I. whereas, type 3 pili expressed by Klebsiella pneumonia unwind at ~67 pN, i.e., almost 10 fold higher than CFA/I.
As reported earlier 3, and also shown in Fig. 3, the n to n+3 interaction of CFA/I compared to P-pili is significantly weaker, which correlates well with the lower unwinding forces measured with FMOT, since the unwinding force is directly connected to the magnitude of the LL interactions that stabilize the quaternary helix-like structure 3. However, the findings in this work indicate that the limited surface area for the n to n+3 interaction is not sufficient to account for the significantly lower unwinding force in comparison with that of P-pili. Comparison of the contact area data of CFA/I and P-pili LL interactions predicted that ~60 % of force was needed to unwind a CFA/I pilus as compared to unwinding of a P-pilus. Nevertheless, the measured unwinding force in situ, with FMOT, was ~3.7 fold smaller. To account for the large apparent discrepancy the results suggests that the number of subunit-subunit interactions and the pitch of the subunits both having a large influence on how much axial force a pilus can withstand.
We therefore propose that the very number of residue-residue interactions and the subunit orientation relative the tensile stress are contributing factors for the reduced force needed to unwind CFA/I pilus filaments, as compared to P-pili and Hib pili. That is, the P-pilus withstands stronger external forces before unwinding, due to its increased LL interactions (surface contact area of ~1150 Å2, including 28 residue-residue interactions) and, to a lesser degree, from the 13° pitch of the subunits from horizontal, as compared to the 4° subunit pitch and very limited contact area (~710 Å2, including 7 residue-residue interactions) for CFA/I pili. These data are consistent with all presently available results from unwinding experiments performed with optical tweezers. This study also shows the benefit of using single molecule force spectroscopy techniques, such as FMOT, as a complement to the static data obtained from e.g., crystallographic and EM images, to gain valuable information otherwise not accessible.
The unwinding force for CFA/I, is not only much lower than for helix-like pili expressed by UPEC P-pili and S-pili, the peak to peak value of the force is also higher. There are two possible explanations for the high fluctuations in the qualitatively constant force measured throughout Region II. First, when a subunit-subunit bond opens/closes, the instantaneous force change measured by the FMOT depends on the increase in length, ΔxAB, from the bond opening, and the force subsequently returns to its steady-state value. That is, the measured peak-to-steady-state force will increase with increased elongation distance per bond opening. Therefore, if the opening of a CFA/I subunit results in a larger momentary elongation change than ΔxAB seen for P, S, and type 1 pili, the average fluctuations in force would consequently be larger. Second, the opening/closing rates of the subunit-subunit bonds are correlated with the extent of LL interactions, with weaker interactions giving rise to faster opening/closure rates. If several subunits open during the time scale of the experiment, they would be registered as a single event, which would result in larger fluctuations in the measured force. Data from EM show weak LL interactions for CFA/I pili and smaller contact surface area between layers, so that the second explanation, increased frequency of subunit-subunit bonds opening, is expected to be the major mechanism underlying the large force fluctuations.
FMOT allows free rotation of the tethered object during force measurements. The multiple peptide bonds in the hinge region between subunits of P-pili could, therefore, rotate during the extension in region II. CFA/I pili, with no linker region between subunits, is more sterically constrained, and may require a proline isomerization for rotation to a linear fiber 3. Such an isomerization can be conformationally induced in the absence of an enzyme to catalyze this reaction 21, but the force required is larger than that exerted by FMOT in region II. This inability to rotate individual subunits would account for the extended, kinked helical form of CFA/I observed by EM. In this model, CFA/I pili would remain a kinked helix as it enters region III, with the filament direction not always co-axial with the fibril axis. The FMOT force exerted along the helix axis is distributed into a component that follows the direction of the filament, and an axial component. It is only the force component in the direction of the filament that is available to break the H-bonds of the beta strand/beta strand interaction, and thereby slide the N-terminal extension of the n+1 subunit out of the groove of the n subunit. Therefore, an extended but kinked fibril such as CFA./I as it enters region III, would require a stronger force to break apart a CFA/I pilus than would be required to break a P-pilus.
At the end of region II, there is sometimes a dip of 2 pN in the force prior to its linear increase in region III, data not shown. We propose that this dip represents a rotation of the tethering bead in the trap. That is, in some cases a twist is accumulated in the CFA/I pilus as the layer-to-layer bonds break but the subunits do not turn. At the end of the unwinding region, the tethering bead rotates in the trap, to reduce the stored energy just as the pilus is entering region III of the force curve.
The increasing force seen in region III is an effect of the inherent resistance of the open helical structure to be linearized. In this region of the force-extension curves of P, S, and type 1 pili, all showed an S-shaped curve. This pseudo-elastic, non-linear force-extension has been attributed to a conformational change of individual subunits at a force interval of 45-70 pN, and this response perfectly matches the sticky-chain theory for linearized helix-like pili 20. Polymers that meet the criteria of a “sticky chain” are those comprised of units weakly connected by joints that are altered by force in an individual or cooperative manner 22. A pilus under stress satisfies these criteria. For P-pili in region III, a conformational change of individual subunits is clearly apparent from the S-shape of the curve in this region 12. CFA/I pili, however, do not show such a conformational change for forces up to 70 pN, providing evidence that CFA/I pili maintain a stable structure at high forces, with both a persistence length that correlates with the largest measured resistance to bending and a short N-terminal extension of CfaB pilins. This stability of the subunit structure occurs despite the much weaker LL bonds that hold together the helix-like structure of the wound pilus filament.
Persistence length measurements show that CFA/I pili are, of those pili measured to date, the most easily bent when in their helical rod form, and yet are the most rigid when they are unwound into their extended, fibrillar conformation. High flexibility of the helical structure could be of advantage for two reasons. First, a flexible pilus can “search”, due to thermal fluctuations, for receptors in a larger volume than a stiff pilus, thus increasing the probability for adherence. Secondly, high flexibility allows for a more uniform distribution of the shear forces exposed to a bacterium than if the pilus were stiff. That could be of advantage in an environment where the flow tends to include back and forth motion, which exposes a bacterium to normal forces that will apply a pressure in the axial direction of a pilus. It was shown in a study by Jeffrey et al. that the peristaltic motion in the ileum creates both shear forces, including reversal wall shear forces, and normal forces 2. A pilus with large degree of flexibility will thereby be able to bend with the flow and absorb the axial pressure by bending the structure without breaking an LL interaction.
The adhesins expressed by CFA/I and type 1 pili are similar in tertiary structure and show shear enhanced binding 23. In a recent study it was shown that a CFA/I pilus manifests shear-enhanced binding to its erythrocyte receptors in a stepwise increase in shear up to 5.0 dynes cm−2 23. This value is in the middle of the 1-10 dynes cm−2 range of shear forces originating from the peristaltic activity in the gastrointestinal tract 2. The unwinding and kinetic data determined for both CFA/I and type 1 pili open up an interesting study and discussion of why the adhesins of these pili both show shear enhanced binding, when at the same time, the rod exhibits a 4-fold difference in unwinding force and ~250 times difference in kinetics.
Findings in this paper, through assessment of data from multiple experimental techniques, all point to CFA/I pili having the absolutely weakest LL bonds of all helix-like pili investigated so far. Compared to the P-, S-, and type 1, the extension speed at which CFA/I pili transition from steady-state to dynamic unwinding is ~4 times higher than P-pili, ~2-7 times higher than S pili, and ~250 times higher than type 1 pili (see reference 8 for a comprehensive table). Although type 1 pili are found on bacteria that colonize many different environments, P-pili are more commonly found on isolates of the upper urinary tract, and type 1 pili on bacteria from the lower urinary tract. The environmental conditions in these regions are different; in the upper urinary tract the urine is transported in boluses via a peristaltic activity, whereas in the lower tract urine is expelled from the bladder via the urethra in a more continuous manner 24; 25. Thus, different environmental conditions in the urinary tract also require dissimilar types of adhesins and rods for optimized bacterial adherence. Since the environmental conditions in the gut are very different compared to any portion of the urinary tract, it is plausible to state that the reduced unwinding force and the fast kinetics of the CFA/I pili have evolved in an optimal fashion for adhesion of the ETEC bacteria.
The E. coli strain HMG11/pNTP119 26 express 0.5-1 μm CFA/I pili on its cell surface as recorded by atomic force microscopy shown in Fig. 1. Bacteria were grown on CFA agar plates at 37°C overnight. Prior to an experiment, a colony from the agar plate was harvested and suspended in 500 μl filtered phosphate buffer solution (PBS, 10mM phosphate, 130 mM NaCl, pH 7.4, Sigma-Aldrich).
Substrates were prepared by adding a 250 μl solution of 9.7 μm polystyrene beads (Duke Scientific Corp., Palo Alto, CA) diluted with filtered MilliQ H2O onto cover slips that were then placed in an oven for 60 minutes at 60° C. The beads were immobilized to the surface and later functionalized with 2 μg / slide poly-L-lysine (Sigma-Aldrich) and worked thereby as mounts for bacteria during a FMOT measurement to prevent optical damage of the bacteria by direct contact with the laser beam. This functionalization creates strong electrostatic bonds with the bacterium and ensures that the bacterium is immobilized during a pilus force-extension experiment. A 25 μl droplet containing bacteria and 3.0 μm polystyrene beads (Duke Scientific Corp., Palo Alto, CA) used as force transducers was added on top of the immobilized beads. Finally, the chamber was closed by adding a top cover slide and placed in a custom made sample holder in the microscope. The experiments were conducted at 25° C.
The optical tweezers system and measurement procedure have previously been described in 15; 27, and the FMOT studies were performed by the same method as those carried out on P, S, and type 1 pili 8; 12. In a typical experiment, a bacterium was trapped and mounted on the 9.7 μm bead at low laser power to prevent optical damage 28. A 3.0 μm bead was subsequently trapped at high laser power and the trapping constant, derived by the power spectrum method 29, was in general ~120 pN/μm. The extension/retraction velocity for steady-state measurements was kept at 0.1 μm/s, whereas dynamic measurements of region II were conducted at 0.1, 1, 2, 4, 8, 16, and 32 μm/s. All data was compensated for Stokes drag force and Faxens law was used for adjustment of trapping near a surface, ~5 μm.
In this work, we focus on exploring the biomechanical properties of the rod. Therefore, in order to avoid any effect of the distal tip and the adhesin, we bound a pilus non-specifically to a bead by the method described in 15. The presented force-extension data originate from a total of 88 measurements from 88 distinct pili. The intervals for the values of the unwinding forces and the persistence lengths correspond to the standard deviations of the statistical data sets, respectively.
Subunit orientations were determined from the three-dimensional structures of CFA/I pili, P-pili, and Hib pili, with their respective major subunits (CfaB, PapA) or homologous subunit (HifA) fitted as published previously 4; 16; 30 ENREF 15 into the maps; for convenience, the fit of CfaB into the CFA/I pilus structure is shown in the Supplemental figure. Subunit-subunit contacts were determined after fitting of the CfaB subunit (pdb 3F84) into the CFA/I pili helical reconstruction map, or the PapA homology-modeled and optimized subunit 30 into the P-pili helical reconstruction map. Contact criteria were ≥ −0.4 Å van der Waal's overlap, and contact area and specific residue-residue interactions were calculated using UCSF Chimera software 31.
Bacteria expressing CFA/I pili were pelleted and resuspended in 3 ml PBS per gram of wet cell pellet weight. Pili were heat-extracted at 65°C for 25 min, and cells were removed by centrifugation at 10,000 × g for 30 min. Ammonium sulfate was added to the supernatant, 0.24 g per ml, and rocked for a minimum of 2 hours at room temperature. Precipitated pili were pelleted by centrifugation at 12,000 ×g, resuspended in PBS, and the ammonium sulfate precipitation procedure was repeated. The resuspended pili were then dialyzed against TE (10 mM Tris pH 7.4 with 0.1 mM EDTA).
For imaging, CFA/I pili were placed on glow-discharged, carbon-coated grids, washed with TE, and stained with 1% uranyl acetate. Images were recorded at 120 kV in a Philips CM12 electron microscope on Kodak SO163 film, and scanned on a Nikon 9000 scanner.
We used a Monte Carlo method to stochastically simulate the force-extension behaviour of a single pilus. The method is built on the Bell-Evans theory for bond kinetics in combination with a WLC model, similar to the one described in refs. 18; 32. In short, the Bell-Evans theory states that the probability for a bond transition from state A to state B is described by the off-rate, koff, as
where is the thermal off-rate, f is the force applied to the bond, kB is Boltzmann's constant and T is the absolute temperature. The applied force thus changes the bond transition rate that is equivalent, in our case, to the unwinding rate of the individual subunits. The force-extension relation is described by an elastic model in combination with a WLC that is commonly used to describe macromolecules in thermodynamic equilibrium. In the WLC the force is coupled to the end to end length of the unwound part of the pilus as
where p is the persistence length, and Lc is the contour length of the unwound part of the pilus, respectively.
AFM imaging of bacteria with pili was done essentially as described earlier with some modifications 33. Bacterial cells were suspended 50 μl in filtered water before 10 μl were placed onto freshly cleaved ruby red mica (Goodfellow Cambridge Ltd, Cambridge). The cells were incubated for 5 minutes at room temperature and blotted dry before being placed into a desiccator for a minimum of 2 hours. Images were collected with a Nanoscope V Multimode AFM equipment (Veeco software) using TappingModeTM with standard silicon cantilevers oscillated at resonant frequency (270-305 kHz). Images were collected in air at a scan rate of approximately 0.5-1.5 Hz.
The crystal structure of CfaB was fit into the density map from a 3-dimensional reconstruction of isolated and purified CFA/I pili and the helical symmetry determined by EM was used to propagate the fit (Li et al. 2009; Structure of CFA/I fimbriae from enterotoxigenic Escherichia coli. Proceedings of the National Academy of Sciences of the United States of America 106, 10793-10798). This fit of CfaB into CFA/I pilus is shown in side view (A) and 1.5 nm thick cross-sections (B), with a ribbon structure of CfaB and the density map as a surface with mesh superposed. To show the fit in more detail, the EM map is shown as a volume rendering in panel C. Magnification bar, 2.5 nm.
This work was supported by the Young Researcher Award (Karriärbidrag) from Umeå University (to MA) and by grants from the Swedish Research Council (to BEU) and NIH GM055722 (to EB). We are grateful to Mrs. Monica Persson for excellent technical assistance, and to Dr. Stephen Savarino and Ms. Annette McVeigh for development of the CFA/I pili isolation protocol.
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